The goal of this project is to understand how motors which power fast axonal transport promote movement. An important current question is how kinesin or dynein is organized on the organelle surface and microtubule substrate. A scanning transmission electron microscope (STEM) (as described in Project #ZOl NS 02610-09 LN), has allowed kinesin to be visualized directly on the surfaces of microtubules and organelles. Maps of kinesin bound to purified, taxol-stabilized bovine microtubules provided the first direct evidence for cross-bridging of microtubules by single kinesins which suggests that kinesins in cells might also translocate microtubules and therefore have some role in slow as well as fast axonal transport. These observations are now being extended to organelles, where we have recently been able to separate anterograde, kinesin-powered organelles, from retrograde, dynein-powered organelles, and to endoplasmic reticulum (ER) where we have shown incidentally that the ER makes one continuous system throughout the Purkinje neuron. The dynamic properties of other biological motors are being studied for comparison with the axonal transport motors; the bacterial flagellar motor in E. coli has also been shown to depend on interactions of the flagellar structure with membrane proteins although these motors are driven by rather than controlled by ionic gradients. This motor system, like the axonal motor system, can also switch direction of translocation. A newly discovered structural component of the flagellar motor has led us to propose a novel structural model. Molecular genetic analysis of the new structural components is expected to lead to an understanding of the directional switching.